Tetrameric structure of mitochondrially bound rat brain hexokinase: a crosslinking study.
Rat brain hexokinase (ATP:D-hexose-6-phosphotransferase; EC 2.7.1.1) was derivatized with sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)ethyl-1,3'-dithiopro pionate (SAND), a photosensitive and cleavable crosslinking agent. The catalytic activity and mitochondrial binding properties of the enzyme were only marginally affected by reaction with SAND. When the derivatized enzyme was bound to liver mitochondria, photolysis resulted in extensive formation of a single crosslinked species with estimated molecular mass 460 kDa. This was determined to contain only hexokinase and thus represents a tetramer of the 116 kDa (apparent molecular mass in gel system used) monomeric enzyme. Although small amounts of tetramer were detected after photolysis of relatively high concentrations of derivatized enzyme in free solution, tetramer formation was greatly enhanced when the enzyme was bound to mitochondria. No evidence of dimeric or trimeric structures was seen even when only a small fraction of the available binding sites on the mitochondrial membrane were occupied. It is thus concluded that tetramer formation is closely linked with binding of the enzyme to the outer mitochondrial membrane and, more specifically, to the pore structure through which metabolites traverse this membrane. It is speculated that a tetrameric structure surrounding the mitochondrial pores may facilitate interactions between the hexokinase reaction and oxidative phosphorylation, mediated by the adenine nucleotides which are common intermediates in these reactions.
Xie
G
G
Department of Biochemistry, Michigan State University, East Lansing 48824.
Wilson
J E
JE
D000818:Animals; D001386:Azides / Q000378:metabolism*; D001921:Brain / Q000201:enzymology*; D003432:Cross-Linking Reagents / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006593:Hexokinase / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D046911:Macromolecular Substances; D008928:Mitochondria / Q000201:enzymology*; D008930:Mitochondria, Liver / Q000201:enzymology; D008970:Molecular Weight; D011485:Protein Binding; D051381:Rats; D013388:Succinimides / Q000378:metabolism*
D001386:Azides; D003432:Cross-Linking Reagents; D008024:Ligands; D046911:Macromolecular Substances; D013388:Succinimides; C062493:sulfosuccinimidyl-2-(3-azido-2-nitrobenzamido)ethyl-1,3'-dithiopropionate; D006593:Hexokinase